Induction of Ferroptosis in Glioblastoma and Ovarian Cancers by a New Pyrrole Tubulin Assembly Inhibitor

We synthesized new aroyl diheterocyclic pyrrole (ARDHEP) 15 that exhibited the hallmarks of ferroptosis. Compound 15 strongly inhibited U-87 MG, OVCAR-3, and MCF-7 cancer cells, induced an increase of cleaved PARP, but was not toxic for normal human primary T lymphocytes at 0.1 μM. Analysis of the levels of lactoperoxidase, malondialdehyde, lactic acid, total glutathione, and ATP suggested that the in vivo inhibition of cancer cell proliferation by 15 went through stimulation of oxidative stress injury and Fe2+ accumulation. Quantitative polymerase chain reaction analysis of the mRNA expression in U-87 MG and SKOV-3 tumor tissues from 15-treated mice showed the presence of Ptgs2/Nfe2l2/Sat1/Akr1c1/Gpx4 genes correlated with ferroptosis in both groups. Immunofluorescence staining revealed significantly lower expressions of proteins Ki67, CD31, and ferroptosis negative regulation proteins glutathione peroxidase 4 (GPX4) and FTH1. Compound 15 was found to be metabolically stable when incubated with human liver microsomes.


■ INTRODUCTION
Glioblastoma multiforme (GBM), a grade IV glioma and one of the most aggressive forms of malignancy, arises within the brain and infiltrates rapidly into adjacent brain tissue. 1,2 GBM represents 45% of malignant tumors of the brain and the central nervous system. 3 The poor prognosis persists following standard treatments with surgery, radiotherapy, and chemotherapy. Patients with GBM tumors have only a 14 month life expectancy from diagnosis and are difficult to treat. They tend to relapse and show drug resistance to current therapy. 2,3 Standard chemotherapy of GBM includes temozolomide, a mustard that does significantly increase survival when combined with radiotherapy. Bevacizumab, an anti-VEGF monoclonal antibody (mAb), was approved for the treatment of recurrent GBM. Carmustine is a nitrosourea in use for the treatment of both recurrent and newly diagnosed GBM, but it causes severe bone marrow, liver, and kidney toxicity. 4,5 Ovarian cancer (OC) is the most frequent cause of death among gynecologic cancers and the deadliest malignancy in women. NIH cancer statistics for 2021 estimated 21,410 new cases (1.1% of all new cancer cases) and 13,770 deaths. The 5 year relative survival for 2011−2017 is 49%. 6 Debulking surgery and radiation therapy are the standard treatment for nonmetastatic disease. Depending on the type of OC, different therapeutic managements are used. Chemotherapy with tubulin binding agents, mustards, and intercalating agents are standard components of OC treatment. Advanced level treatment options may also include targeted therapy, immunotherapy, and hormone therapy. 7,8 After the first-line treatment, GMB tumors progress with limited treatment options. Current systemic therapy with temozolomide and nitrosoureas has limited efficacy, and resurgery or re-irradiation may be useful in selected cases. Positive therapeutic responses to recurrent GMB can be observed in a highly selected and very limited patient population. 9 Standard chemotherapy of recurrent OC with tubulin binding/platinum agents significantly improved progression-free survival up to 15 months after the introduction of the mAb bevacizumab. 10 However, nearly 23% of patients relapse within 6 months after the end of primary chemotherapy and 60% relapse after a further 6 months. 11,12 Bevacizumab also has been used extensively to treat recurrent GMB in patients who have failed the first line therapy. 13 However, despite a high initial response rate, the effect is transient and the tumors of most patients progress rapidly. 14,15 Apoptosis (programmed cell death) has been long recognized as critical for sustained tumor suppression following anticancer treatments. Recently, ferroptosis is being increasingly investigated as nonapoptotic, iron-dependent regulated cell death (RCD), distinct from other forms, such as necroptosis, pyroptosis, and alkaliptosis, with potential to overcome the block of apoptosis in some cancer cells. 16,17 The ferroptotic pathway is mainly triggered by peroxidation of extra-mitochondrial lipid arising from the accumulation of iron-dependent reactive oxygen species (ROS) The induction of ROS production in most tumors follows a state of high oxidative stress caused by excessive iron derived from abnormalities of two major redox systems, lipid peroxidation and thiols, and from aberrant iron metabolism. 18 Many cancers are ferroptosis-related. 19 Therefore, inducing ferroptosis can be an effective approach to eradicate residual or resistant cancer cells. 20 Much evidence also supports the idea of inducing ferroptosis in GBM therapies. 21 Moreover, OC cells have shown susceptibility to ferroptosis because excess iron can overload tumor-initiating cells following overexpression of transferrin receptor 1 and a decrease in the level of the iron efflux pump ferroportin. 22 In this work, we replaced the 1-(methylphenyl) group of 1 23 with a pyridine or pyrimidine ring and kept fixed the hydrogen, phenyl, or furan-2-yl moiety at position 4 of the pyrrole. The latter heterocyclic ring has shown a tight interaction with the colchicine site of tubulin. 24 We found that a new aroyl diheterocyclic pyrrole (ARDHEP) derivative, 15, whose mechanism is the inhibition of tubulin polymerization, exhibited the hallmarks of ferroptosis rather than the conventional apoptosis found with 1 (Chart 1). However, angiogenic effects were similar for both compounds. In this paper, we report the synthesis of 15 and its antitumor activity in vitro and in vivo, as well as its selectivity for cancer cells as compared with normal human cells.   24 Compounds 18 and 19 were prepared from 1phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one or 3-(furan-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one and p-toluenesulfonylmethyl isocyanide in dimethyl sulfoxide/ diethyl ether, respectively, in the presence of sodium hydride, as previously described. 23 Biology. Inhibition of Cancer Cell Growth. MCF-7 Cells. Inhibition of the growth of human breast carcinoma (MCF-7) cancer cells was strongly correlated with the nature of the heterocycle at position 1 of the pyrrole and the R 2 substituent at the 4 position (Table 1). Compounds 2−5 without the R 2 aromatic substituent were weak inhibitors of MCF-7 cancer cell growth, independent of the heterocycle at position 1 of the pyrrole nucleus. Among pyridines 6−11, pyridin-2-yl 6 and 9 and pyridin-3-yl 7 and 10 derivatives inhibited cell growth with IC 50 's of 18−50 nM, while pyridin-4-yl compounds 8 and 11 were weak inhibitors. Among compounds 13−16, pyrimidin-2yl 13 and 15 and pyrazin-2-yl 12, derivatives with nitrogen atom(s) close to the carbon atom linked to the pyrrole nitrogen, showed strong inhibition of MCF-7 cell growth, while pyrimidin-5-yl derivatives 14 and 16 were poor inhibitors of cell growth. Introduction of the furan-2-yl at position 4 of the pyrrole tended to reinforce the inhibition of MCF-7 cell growth; in particular, derivative 15 exhibited the strongest inhibition within the series with an IC 50  viability of human GBM U-87 MG cells with an IC 50 of 10.06 nM and ovarian adenocarcinoma OVCAR-3 cells with an IC 50 of 2.85 nM. Compound 15 was thus 3.5-fold less effective as an inhibitor of the U-87 MG cells as compared to the OVCAR-3 cell line ( Figure 1A,B). To determine if the drug caused programmed cell death, we analyzed the cleavage of PARP by immunoblotting. As shown in Figure 1B, right panel, compound 15 induced a significant increase of cleaved PARP, starting from 5 nM. The proliferation of GBM cells U-87 MG was also inhibited by the same compound in a dosedependent fashion, and this, too, was accompanied by the PARP cleavage, starting from 20 nM, consistent with their lower sensitivity to compound 15 as compared with the OVCAR-3 cells.
Human Primary T Lymphocytes. Potential toxicity on healthy cells was evaluated by treating human primary T lymphocytes with 0.1, 1.0, and 2.5 μM 15 or with control vehicle (dimethyl sulfoxide; DMSO). The frequency of early and late apoptotic cells was assayed by staining with annexin V and propidium iodide for 48 or 72 h. Compared to untreated or DMSO control, flow cytometric analysis showed that exposure to 0.1 μM 15 did not change cell viability, while some cytotoxicity was observed with 15 at 1.0 or 2.5 μM. These results indicated that this compound at 0.1 μM is not toxic for normal cells (Figures S1 and S2  Biochemical analysis showed that levels of lactoperoxidase (LPO), a heme-containing mammalian peroxidase, malondialdehyde (MDA), an end product of lipid peroxidation, and lactic acid in tumor tissues derived from the 15-treated groups were appreciably higher than those in the control groups ( Figure 5, bottom panel). However, the levels of total glutathione (T-GSH) [glutathione (GSH) + oxidized glutathione (GSSG)] and ATP were significantly lower in tumor tissues derived from the 15-treated groups than those in the control groups ( Figure 5, bottom panel).
Cell death in GSH-depleted cells has been described as occurring through ferroptosis and autophagy and that ferroptosis is a primary mechanism of GSH depletion-induced cell death in retinal pigment epithelial cells. 25 We also found   that the concentrations of Fe 2+ in tumor tissues derived from the 15 treatment groups were significantly higher than those in the control groups ( Figure 5). The presence of sufficient free intracellular iron and the presence of membrane oxidizable phospholipids acylated with polyunsaturated fatty acids are both prerequisites for the occurrence of ferroptosis. 26 Thus, our results are consistent with the conclusion that because 15 caused oxidative stress injury and Fe 2+ accumulation, in vivo inhibition of proliferation of the cancer cells was caused by ferroptosis.
Gene Expression Profiles of Proliferation and Ferroptosis in Tumor Tissues by Compound 15. Quantitative polymerase chain reaction (qPCR) technique was used to analyze mRNA expression profiles involving ferroptosis, cell proliferation, and cell death (total 69 genes) in tumor tissues of the 15-treated and control groups ( Figure 6, top and bottom panels).
According to the 2 −ΔΔCt method, 27 significance was calculated as follows: A value ≥1.5 was considered significant for indicating an increased expression level. Accordingly, the expression levels of 25 genes in U-87 MG tumor tissues derived from the 15treated group were significantly elevated compared to those in the control group ( Figure 7, top panel). The expression levels of 23 genes in SKOV-3 tumor tissues derived from the 15treated group were significantly elevated compared to the control group (Figure 7, bottom panel). Eleven genes (Ptgs2, Gls2, Nfe2l2, Sat1, Akr1c1, Hspb5, Gpx4, Tf rc, Cbs, Nrf 2, and Cisd1) were present in both groups ( Figure 8).
Using the protein−protein interaction prediction tool STRING v11, 28,29 we analyzed the protein network of the above 11 genes related to ferroptosis. Among them, the results suggested that the Ptgs2/Nfe2l2/Sat1/Akr1c1/Gpx4 genes have potential to regulate protein−protein interactions ( Figure  9): the Ptgs2 gene can cause angiogenesis, differentiation, and promotion of cancer through dysregulation of COX-2; 30 the Nfe2l2 gene encodes for NRF2 that in humans is a central regulator of redox, metabolic, and protein homeostasis; 31 the Sat1 gene encodes for an acetyltransferase that is involved in the regulation of the intracellular concentration of polyamines and their transport out of cells; 32 the Akr1c1 gene encodes for the AKR1C1 enzyme that catalyzes the reduction of aldehydes and ketones to their corresponding alcohol and is overexpressed in the lungs, ovary, uterine cervix, skin, and colon carcinomas; 33 the GPX4 gene encodes for the enzyme GPX4,  which protects cells against membrane lipid peroxidation. GPX4 is an essential regulator of ferroptotic cancer cell death. 34 A graph from Gene Ontology (GO) analysis 35 ( Figure  10) shows that 15 could affect molecular functions, such as protein binding, protein dimerization activity, and oxidoreductase activity.
Immunofluorescence staining showed that the tumor tissues derived from the 15-treated groups have significantly lower expression of Ki67 and CD31 and of ferroptosis negative regulation proteins GPX4 and FTH1. The proliferation marker Ki67 is a nuclear protein that is strongly associated with tumor cell proliferation and is an established prognostic indicator for cancer assessment by biopsy. 36 CD31 is a well-defined marker of angiogenesis, along with the vascular endothelial growth factor (VEGF). 37 CD31 is highly expressed on the surface of endothelial cells, and CD31 is involved in angiogenesis in early breast cancer. 38 GPX4 and FTH1 are ferroptosis negative regulation proteins. The selenoenzyme GPX4 reduces membrane phospholipid hydroperoxides and maintains cellular redox homeostasis, using glutathione (GSH) as a cofactor. 39 Inactivation or depletion of GPX4 in a variety of cell types can induce ferroptosis. 34 FTH1 has ferroxidase activity, which specifically oxidizes ferrous iron (FeII) to ferric iron (FeIII). FTH1 regulates angiogenesis during inflammation and malignancy, interacts with several signaling elements involved in critical cellular pathways, and activates p53 under oxidative stress. FTH1 acts as a tumor suppressor in nonsmall cell lungs and breast and ovarian cancers, as well as as a tumor promoter in metastatic melanoma cells. 40 In summary, we conclude that 15 promoted ferroptosis in tumor tissues by stimulating the ferroptosis protein regulation network ( Figure 11).
Metabolic Stability. Compound 15 was assessed for its metabolic stability to phase I oxidative metabolism using mouse and human liver microsomes (Table 2), with 7ethoxycoumarin (7-EC) and propranolol as control compounds. Compound 15 was highly metabolized after incubation with mouse liver microsomes, showing a very high intrinsic clearance value of 461 μL/min/mg protein. However, compound 15 was found to be much more metabolically stable when incubated with human liver microsomes showing a medium intrinsic clearance of 36 μL/ min/mg protein (Table 3). Liquid chromatography tandem mass spectroscopy (LC−MS/MS) analyses were carried out using an ESI(+) interface in multiple reaction monitoring (MRM) mode. Conditions and MRM transitions applied to the compounds are described in Table 4. The metabolic stability profile of 15 did not differ much from propranolol.
From a drug development point of view, the stability shown in the presence of human liver microsome enzymes may represent a good starting point for compound optimization.
Druglike Properties. The ADME profile of 15 was predicted through representative descriptors by the SwissADME web site 44 (Table 5). According to the metabolic stability data reported in Table 2, compound 15 was predicted to be the substrate of the highly expressed CYP450 2C9, 2D6, and 3A4 isoforms. 45 The compound does not violate the Lipinski 46 55,56 The association of tubulin with VDAC induces highly voltage-sensitive reversible blockage of the single channel and hampers the cellular metabolism. 57 The microtubule-targeting agents, by binding directly to the tubulin associated with VDAC, cause depolarization of the mitochondrial membrane and interferes with mitochondrial function. 58,59 By interaction with the VDAC, the ferroptosis inducer erastin causes ferroptotic (nonapoptotic) cell death and abnormal functioning of mitochondria. 60,61 We hypothesize that the novel tubulin polymerization inhibitor 15 may interact with tubulin-associated-VDAC, thereby interfering with the function of the mitochondrial activity. The interaction between tubulin and VDAC has been reported as novel target for inducing ferroptosis in cancer cells. 62 Compound 15 was metabolically stable when incubated with human liver microsomes and showed a medium intrinsic clearance of 36 μL/min/mg protein. In summary, we described the synthesis and antitumor activities in vitro and in vivo of a tubulin polymerization inhibitor, compound 15, that induced cell death and presented the typical hallmarks of ferroptosis rather than conventional apoptosis. The biological profile of 15, together with its stability in the presence of human liver microsome enzymes, highlights compound 15 as a robust lead compound for further optimization to provide new anticancer drugs based on alternative mechanisms of action.
■ EXPERIMENTAL SECTION Chemistry. All reagents and solvents were handled according to the material safety data sheet of the supplier and were used as purchased without further purification. MW-assisted reactions were performed on a CEM Discover SP single-mode reactor equipped with an Explorer 72 autosampler, controlling the instrument settings by PC-running CEM Synergy 1.60 software. Closed vessel experiments were carried out in capped MW-dedicated vials (10 mL) with a cylindrical stirring bar (length 8 mm, diameter 3 mm). Stirring, temperature, irradiation power, maximum pressure (Pmax), pressure set point, times at set point, delta pressure, PowerMAX (simultaneous cooling-while-heating), ActiVent (simultaneous venting-while-heating), and ramp and hold times were set as indicated. Reaction temperature was monitored by an external CEM fiber optic temperature sensor. After completion of the reaction, the mixture was cooled to 25°C via air-jet cooling. Organic solutions were dried over anhydrous sodium sulfate. Evaporation of solvents was carried out on a Buchi Rotavapor R-210 equipped with a Buchi V-850 vacuum controller and a Buchi V-700 vacuum pump. Column chromatography was performed on columns packed with silica gel from Macherey-Nagel (70−230 mesh). Silica gel thin layer chromatography (TLC) cards from Macherey-Nagel (silica gelprecoated aluminum cards with fluorescent indicator visualizable at 254 nm) were used for TLC. Developed plates were visualized with a Spectroline ENF 260C/FE UV apparatus. Melting points (m.p.) were determined on a Stuart Scientific SMP1 apparatus and are uncorrected. Infrared (IR) spectra were recorded on a PerkinElmer Spectrum 100 Fourier transform-IR (FT-IR) spectrophotometer equipped with a universal attenuated total reflectance accessory and    IR data acquired and processed by PerkinElmer Spectrum 10.03.00.0069 software. Band positions and absorption ranges are given in cm −1 . Proton nuclear magnetic resonance (1H NMR) spectra were recorded with a Bruker Avance (400 MHz) spectrometer in the indicated solvent, and the corresponding fid files were processed by MestreLab Research SL MestreReNova 6.2.1−769 software. Chemical shifts are expressed in δ units (ppm) from tetramethylsilane. Compound purity was checked by high-pressure liquid chromatography (HPLC). Purity of tested compounds was found to be >95%. The HPLC system used (Thermo Fisher Scientific Inc. Dionex UltiMate 3000) consisted of an SR-3000 solvent rack, an LPG-3400SD quaternary analytical pump, a TCC-3000SD column compartment, a DAD-3000 diode array detector, and an analytical manual injection valve with a 20 μL loop. Samples were dissolved in acetonitrile (1 mg/mL). HPLC analysis was performed by using a Thermo Fisher Scientific Inc. An Acclaim 120 C18 column (5 μm, 4.6 mm × 250 mm) at 25 ± 1°C, with an appropriate solvent gradient (acetonitrile/water), flow rate of 1.0 mL/min, and a signal detector at 206, 230, 254, and 365 nm were used. Chromatographic data were acquired and processed by Thermo Fisher Scientific Inc. Chromeleon 6.80 SR15 Build 4656 software. Ultrahigh-performance liquid chromatography (UHPLC) experiments were carried out on an Accela UHPLC System Thermo Fisher Scientific (San Jose, CA), which consisted of an Accela 1250 Pump, an Accela autosampler, and an Accela PDA photodiode array detector. Chromatographic data were collected and processed using the Thermo Xcalibur Chromatography Manager software, version 1.0. A guard cartridge system (SecurityGuard Ultra UHPLC) has been connected to an analytical column Kinetex 2.6 μm EVO C18 100 Å 100 × 3.0 mm (L × I.D.), both from Phenomenex, Torrance, CA, USA. All analyses were performed at 30°C, and the mobile phase was filtered through 0.2 μm Omnipore filters (Merck Millipore, Darmstadt, Germany). The mobile phase was delivered at a total flow rate of 0.5 mL/min. The analyses were carried out in elution gradient. Specific mobile phase and gradient are reported for each compound in Figures S3− S17, Supporting Information. Relative areas (%) recorded at 254 nm are shown in Table S1, Supporting Information. Analyses were performed in triplicate. Acetonitrile, methanol, water, and trifluoroacetic acid of HPLC gradient grade were purchased from Sigma Aldrich (St. Louis, MO).
Cell Viability Assays. The methodology for the evaluation of the growth of human MCF-7 breast carcinoma cells was previously described, except that cells were grown for 96 h for IC 50 determinations. 66 U-87 MG and OVCAR-3 proliferation assays were performed plating 10 4 cells/cm 2 in a Multiwell 24. The day after, cells were treated with compound 15, and DMSO was used as a control. Cells were counted 48 h later with a Burker counting chamber, after dilution in TrypanBlue (#T6146 Sigma-Aldrich). IC 50 was calculated by using data from the dose−response curves after 48 h of drug treatment using Graphpad Prism 7.0 software, as previously described. 67 Apoptotic cell death was evaluated using the APC Annexin-V Apoptosis Detection Kit with PI (Thermo Fisher Scientific). Briefly, 1.5 × 10 6 /mL cells were cultured in 48-well plates, untreated or treated with different concentrations of 15 for 48 or 72 h. Cells were then stained using annexin-V/APC and propidium iodide according to the manufacturer's instructions. Cell populations were acquired using a FACS Canto II flow cytometer (BD Biosciences). Flow cytometric analysis was performed using Flow Jo Flow Cytometric Analysis Software.
Western Blotting. Western blotting was performed as previously described 68 by lysing cells in denaturing buffer sodium dodecyl sulfate (SDS)−urea. Extracts were sonicated, their protein quantified, and aliquots were loaded into an SDS−polyacrylamide gel. After electrophoresis, the proteins in the gel were transferred onto a nitrocellulose membrane (cat. no. NBA085C001EA, PerkinElmer), which then was blocked with 5% milk in TBS-T (Tris HCl with 0.1% Tween 20) and incubated with primary antibodies overnight: PARP antibody diluted 1:1000 (cat. no. 9542S CST) and actin antibody diluted 1:10,000 (cat. no. A5441, Sigma Aldrich). The next day, the membrane was extensively washed with TBS-T and incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies diluted in the 5% milk solution. Detection of the HRP signal was performed using ECL (cat. no. K-12045-D50, Advansta).
Statistical Analysis. All experiments were performed multiple times to reach statistical significance, as specified in the figure legends. Statistical analysis was performed using GraphPad Prism version 7.0 for Mac. IC 50 values were calculated by using a nonlinear regression formula, using the percentage of cells number and the logarithmic value of drug concentrations. Data were analyzed with analysis of variance (one-way ANOVA test). Data with *p < 0.05 were considered statistically significant.
In Vivo Xenograft Experiments. Briefly, all 10 week-old female BALB/C nu/nu mice (24 mice) were purchased from the Shanghai University of Traditional Chinese Medicine with Institutional Animal Care and Use Committee approval in accordance with institutional guidelines. All mice were randomly divided into four groups. In #1 group (4 mice), 1 × 10 8 cells/mL from U-87 MG at the logarithmic growth phase were harvested and inoculated subcutaneously, followed by intraperitoneal injection of 100 μL of 15 (25 mg/kg) every 2 days. In #2 group (4 mice), 1 × 10 8 cells/mL from U-87 MG at the logarithmic growth phase were harvested and inoculated subcutaneously, followed by intraperitoneal injection of 100 μL of saline every 2 days. In #3 group (4 mice), 1 × 10 8 cells/mL from SKOV-3 at the logarithmic growth phase were harvested and inoculated subcutaneously, followed by intraperitoneal injection of 100 μL 15 (25 mg/kg) every 2 days. In #4 group (4 mice), 1 × 10 8 cells/mL from SKOV-3 at the logarithmic growth phase were harvested and inoculated subcutaneously, followed by intraperitoneal injection of 100 μL of saline every 2 days. After continuous feeding for 40 days, the mice were sacrificed, and the tumors were removed. The tumors were weighed, and the volumes were calculated using the following formula: Tumor volume (cm 3 ) = (ab 2 )/2 (a: the longest axis (cm), b: the shortest axis (cm)).
HE Staining. Tissue samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The paraffin-embedded tissues were cut into 4 μm sections using a microtome, and the sections were affixed onto glass slides. Subsequently, the sections were dewaxed using xylene and subjected to dehydration in an ethanol gradient. The sections were stained with hematoxylin (H) for 5 min at room temperature, and then, 1% ethanol was added for 30 s for differentiation. Afterward, aqueous ammonia was added for 1 min for blueing, followed by rinsing in distilled water for 5 min. Subsequently, the sections were stained with eosin (E) for 2 min at room temperature and then rinsed with distilled water for 2 min. Then, decolorization over an ethanol gradient was performed, and Journal of Medicinal Chemistry pubs.acs.org/jmc Article xylene was added for 2 min for clearing. Finally, the sections were sealed and mounted with neutral resin. Immunofluorescence Staining. Briefly, fresh tissues were immersed in 4% paraformaldehyde (Sigma-Aldrich) for fixation at room temperature for 30 min. The tissues were then dehydrated in an ethanol gradient, embedded in paraffin, sectioned (thickness: 6 μm), and immersed in xylene for dewaxing. Tissue sections were blocked with immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37°C for 30 min. The blocking solution was then discarded, and the sections were washed three times at room temperature for 5 min, each with immunohistochemical washing solution (Beyotime Biotechnology). Then, primary antibodies (rabbit anti-GPX4 antibody [EPNCIR144] (ab125066), rabbit anti-ferritin heavy chain antibody [EPR18878] (ab183781), and rabbit anti-Ki67 antibody (ab15580), Abcam, MA, USA) were added and incubated at 37°C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Then, the secondary antibody (goat anti-rabbit IgG H&L (Alexa Fluor 488), Abcam, MA, USA) was added, and the tissues were incubated at 37°C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min, each with immunohistochemical washing solution (Beyotime Biotechnology). Finally, immunofluorescence blocking solution (Sigma-Aldrich) was added, and the sections were mounted.
RNA Extraction, RNA Extraction with Reverse Transcription Reaction, and qPCR. According to the instructions of the RNAprep pure Tissue Kit (TIANGEN Biotech (Beijing) China) Co., Ltd., Beijing, China), to about 20 mg of human tissue specimen was added 800 μL of lysis solution, and the mixture was ground, homogenized, and centrifuged. To the supernatant, 200 μL of chloroform was added, and the mixture was mixed by inverting and centrifuged at 4°C at 13,400 × g for 15 min. To the supernatant, two volumes of absolute ethanol were added, mixed by inversion, and centrifuged at 4°C at 13,400 × g for 30 min. Ethanol precipitation and centrifugation of the supernatant were repeated. The RNA pellet was suspended with 500 μL of 75% ethanol and centrifuged at 13,400 × g at 4°C for 5 min. The supernatant was discarded, the excess liquid was removed, and the pellet was dissolved in 300 μL of DECP water. One microliter of the RNA solution was used to measure the ratio of A 260 −A 280 (generally 1.8−2.0) to determine the purity and approximate concentration of the RNA. RNA samples were treated with DNase I (Sigma-Aldrich), requantified, and reverse-transcribed into cDNA using the ReverTra Ace-α First Strand cDNA Synthesis Kit (Toyobo). Quantitative real-time PCR (qPCR) was conducted using a RealPlex4 real-time PCR detection system (Eppendorf, Germany) with SYBR Green Realtime PCR Master Mix (Toyobo). On a quantitative realtime PCR instrument, the following reactions were performed: 95°C for 15 min; 94°C for 20 s; 60°C for 34 s, and the fluorescence value was read. The above reactions were performed for 40 cycles. The qPCR primers were described previously. 69,70 In Vitro Oxidative Metabolic Stability. Intrinsic Clearance in Microsomes. 71 Mouse (Sigma Aldrich, CD-1 male, pooled) and human microsomes (Sigma Aldrich, human, pooled) at 0.5 mg/mL were preincubated with the test compound 15 dissolved in DMSO at 1 μM in phosphate buffer 50 mM, pH 7.4, and 3 mM MgCl 2 for 10 min at 37°C. The reaction was then started by adding the cofactor mixture solution (NADP, glucose-6-phosphate, glucose-6-phosphate dehydrogenase in 2% NaHCO 3 ). Samples were taken at 0, 10, 30, 45, and 60 min and added to acetonitrile to stop the reaction. Samples were then centrifuged, and the supernatant was analyzed by LC−MS/ MS to quantify the amount of the compound. A control sample without the cofactor was always added to check the chemical stability of the test compound. Two reference compounds of known metabolic stability, 7-EC and propranolol, were present in parallel testing. A fixed concentration of verapamil was added in every sample as an internal standard for LC−MS/MS analyses. The percentage of the area of the test compound remaining at the various incubation times was calculated with respect to the area of the compound at time 0 min.